Preparation And Functional Confirmation Of Monoclonal Antibodies Against Porcine Epidemic Diarrhea Virus Nucleocapsid

Porcine epidemic diarrhea is an infectious disease caused by porcine epidemic diarrhea virus(PEDV)characterized by dehydration,diarrhea,and severe atrophy of intestinal villi.Since 2010,PEDV virulent strains reemerged in many pig farms around the world,showing high pathogenicity and up to 100% mortality rate for suckling piglets younger than 1-week-old.The reemergence of the PEDV virulent strains has caused significant economic losses in China and the whole world,which severely threatened the pig industry.It is critically important and urgent to develop effective methods for detection of PEDV infection.The nucleocapsid protein(N)is highly conserved among different PEDV strains and is synthesized in the early infection stage.As a result,PEDV N protein is an ideal target for early detection of PEDV infection.To facilitate the establishment of a series of methods for detecting PEDV infection,monoclonal antibodies(mAbs)against PEDV N protein were prepared in this study and their applications were validated in different immunological assays.Firstly,the N gene was amplified from PEDV LJX/2014 strain by RT-PCR and cloned into pET-30a(+)prokaryotic expression vector.The recombinant plasmid,expressing PEDV N protein,was named pET-N.The pET-N was transformed into BL-21(DE3)competent cells to express N protein.The results demonstrated that the target protein was successfully expressed in both secreting proteins and inclusion bodies.After a serial optimization of expression conditions,a large amount of secreted N protein was finally obtained.The recombinant N protein was purified by nickel column affinity chromatography.The result showed that the high purity of recombinant PEDV N protein was obtained in this study.To generate mAbs against PEDV N protein,the BALB/c mice were immunized subcutaneously with 200 ?g purified protein mixed with ISA15 adjuvant and boosted with the same dose every two weeks.One week after the third immunization,the same amount of N protein was injected intraperitoneally.The mice were sacrificed at 3 days post final boost for preparation of splenocytes suspension.The splenocytes were then fused with mouse myeloma cells(SP2/0)with the assistance of PEG.Hybridoma cells were obtained by enzyme-linked immunosorbent assay(ELISA)screen and subjected to three rounds of subcloning.A total of six hybridoma cell lines stably secreting mAbs against PEDV N protein were eventually obtained.These mAbs were identified by the monoclonal antibody typing kit.The result indicated that the heavy chain of the 6 mAbs was IgG1 subclass while the light chain was a ? chain.The titers of ascites prepared from 6 mAbs were higher than 128000 folds dilution.In addition to using ELISA to detect the reactivity of purified PEDV N protein with mAbs,we also used IFA and flow cytometry to detect the reactivity of Vero-E6 cells infected PEDV LJX/2014 strain with 6 mAbs,while the reactivity of cell lysates with mAbs was detected by Western blotting.The results illustrated that the mAbs prepared in this study was able to detect the PEDV infection using IFA,Western blotting,and flow cytometry.The truncated expression strategy was used to identify the epitopes of the mAbs,and the indirect ELISA method was used to analyze the segments of the epitopes of 6 mAbs.The results indicated that the mAb 3F12 strain recognized the linear epitope VAAKDALKSLGI while the remaining mAbs may recognize conformational epitopes located in the N181-379 segment.In conclusion,six mAbs against PEDV N protein were successfully prepared in this study and their subtypes and affinity were determined.The applications of these mAbs were validated by enzyme-linked immunosorbent assay(ELISA),indirect immunofluorescence assay(IFA),western-blot assay,as well as flow cytometry.The epitopes recognized by these mAbs were identified.